375 results about "Protein purification" patented technology

The present invention relates to a method for the production and purification of a protein of interest, and more particularly, the present invention relates to a fusion protein comprising: a self-aggregating peptide, a linker peptide comprising an amino acid sequence selected from an elastin-like polypeptide (ELP) or a protease propeptide (ProP) and the spacer comprising a cleavage site, and a protein portion of interest, and a method for column-free one-step purification of a target protein by expressing the fusion protein.

The invention relates to the technical field of protein purification, in particular to a biological method for purifying fusion protein through two different affinity tags. Two affinity purification tags and a dissolution promoting tag are perfectly combined, so that soluble expression of recombinant protein is ensured, high-purity target protein can be obtained through a two-step mild and efficient affinity purification process, the function and activity of the protein are ensured, a complex'capture-medium purification-fine purification 'multi-step purification process is avoided, and the method is suitable for industrial production. The purification process is shortened, the purification time is shortened, the purification efficiency is improved, and the method can be used for production and purification of functional proteins.

The invention discloses a glycosyl transferase mutant and an application of the glycosyl transferase mutant in synthesis of rebaudioside. According to the invention, single-point mutation and multi-point mutation are carried out on the basis of a glycosyl transferase amino acid sequence as shown in SEQ ID NO: 1, and a mutant with improved catalytic activity is obtained. The catalytic activity, the substrate specificity and / or the substrate specificity of the glycosyl transferase mutant are / is changed, and the catalytic activity of enzyme to a specific substrate can be remarkably improved by mutation at a specific site. And carrying out induced expression and protein purification on the obtained mutation sequence to obtain the mutant enzyme. The mutant enzyme is used as a catalyst, and UDPG is used as a glycosyl donor, so that the catalytic reaction efficiency of substrates such as stevioside ST, rebaudioside A (RebA) and rebaudioside D (RebD) can be obviously improved. According to the glycosyl transferase UGT76G1 mutant constructed by the research, the catalytic activity of the glycosyl transferase UGT76G1 mutant is improved, and efficient production of rebaudioside M is realized by optimizing a reaction system.

The invention belongs to the technical field of protein purification processes, and particularly relates to a method for separating and purifying alpha-lactalbumin in whey. Comprising the following steps: S1, obtaining skimmed milk; s2, performing microfiltration separation on the skimmed milk obtained in the step S1 to obtain whey protein liquid containing alpha-lactalbumin; s3, loading the whey protein liquid containing alpha-lactalbumin obtained in S2 into a balanced strong cation chromatographic column, and collecting a flow-through liquid to obtain separated and purified alpha-lactalbumin; according to the method, the SP Purose 6XL filler is combined with the specific 10-60mM acetic acid buffer solution with the pH value of 4-6, so that the method is particularly suitable for separating the alpha-lactalbumin from other proteins in the whey, and the alpha-lactalbumin is collected in a flow-through manner, so that large-volume sample loading can be realized, and more importantly, the obtained alpha-lactalbumin is high in yield and purity; in addition, the process is simple in extraction step and free of a tedious elution process, a large amount of time can be saved, the production cost is reduced, and industrial production is facilitated.

The invention discloses a split intein and application thereof, the split intein comprises two independent peptide fragments: (a) an N-terminal fragment, the amino acid sequence of which is as shown in SEQ ID NO.1; (b) a C-terminal fragment or a variant thereof, the amino acid sequence of the C-terminal fragment is as shown in SEQ ID NO.2, and the amino acid sequence of the variant is as shown in SEQ ID NO.3 or SEQ ID NO.4. The split intein has splicing activity and excellent splicing rate, the application of the split intein can be widened, the number of amino acids of the C-terminal fragment or the variant thereof is small, and the split intein has remarkable advantages in protein purification, solid-phase synthesis and the like.

The invention belongs to the field of enzyme engineering, and relates to a glycosyl transferase mutant and application thereof in synthesis of salidroside. On the basis of a glycosyl transferase amino acid sequence as shown in SEQ ID NO: 1, iterative saturated mutation is carried out through a molecular modification technology, and the enzymatic activity and catalytic efficiency of the modified glycosyl transferase mutant are greatly improved. The mutation at a specific site can significantly promote the glycosylation activity of a tyrosol substrate. The mutant enzyme is obtained by carrying out induced expression and protein purification on the obtained mutant strain. The substrate conversion rate of the transformed glycosyl transferase mutant for catalyzing the tyrosol glycosylation reaction can reach 95% or above, the yield reaches 55.0 g / L, and an efficient and green new way is provided for salidroside production.

The invention provides a recombined protein rSp14-3-3 of sarcocystis tenella, an antibody and an ELISA (Enzyme-Linked Immunosorbent Assay) diagnostic kit of the recombined protein rSp14-3-3 of the sarcocystis tenella. The research firstly provides the recombined protein rSp14-3-3 of the sarcocystis tenella. According to the present invention, the recombinant protein rSp14-3-3 is used as a gene fragment, the obtained gene fragment is connected to an expression vector to carry out induced expression and protein purification, the purified recombinant protein rSp14-3-3 is used to coat an ELISA plate, the ELISA detection method for sheep infected with sarcosporidium is established, and the specific monoclonal antibody for the recombinant protein rSp14-3-3 is further provided. All the products can be applied to diagnosis of infection of sarcocystis in samples, water samples, food, environmental samples and the like, and technical support is provided for subsequent immunological diagnosis of sheep sarcocystis by using the protein.

The invention relates to a YaxAB nanopore, a system comprising the nanopore and application of the YaxAB nanopore. The YaxAB nanopore of the present invention has a funnel-shaped three-dimensional structure so that the change in current blocking according to the movement of an analyte captured inside the nanopore can be maximized, thereby enabling very sensitive measurement even of the structure and minute changes in physical and chemical properties (including dynamic changes and the like) of the analyte. And can cause very strong electroosmosis with high cation selectivity, such that various types of analytes can be captured and analyzed without being limited to the type of total charge. In addition, with respect to the nanopore of the present invention, the amino acid sequence of the monomer constituting the nanopore can be adjusted to improve protein purification yield and control an oligomerization mechanism, and thus a YaxAB nanopore having a pore size that cannot be formed by a wild-type monomer can be prepared. Furthermore, with respect to the nanopores of the present invention, YaxAB nanopores of various pore diameters can be secured using one-time purification, and nanopores of desired pore diameters can be selectively secured. Thus, limitations of conventional biological nanopores having fixed pore diameters can be overcome, so that for the YaxAB nanopore of the present invention, the size of the nanopore can be adjusted according to the size of an analyte. Further, the YaxAB nanopores of the present invention have a very wide analyte analysis range of dynamic detection range from small analytes to large analytes, which is caused by a funnel-shaped three-dimensional structure having a wide inlet and a narrow outlet. In addition, the YaxAB nanopore of the present invention has the largest nanopore structure among the hitherto biological nanopores, and a large-size analyte, which cannot be detected by conventional biological nanopores, can be detected and analyzed in a single molecule on the basis of a funnel structure having a large inlet. Furthermore, the present invention has established an optimized composition of a membrane in which YaxAB nanopores can be stably present, and by using a sensor implemented on the basis thereof, it is possible to qualitatively and quantitatively analyze various types of analytes having relatively various sizes and charges at a single molecule level with very high resolution and sensitivity.

The present invention provide novel immunofiber compositions for protein or peptide purification and simple and cost-efficient methods and systems using these compositions. In some embodiments, the immunofibers comprise a customized Z-33 peptide derived from Staphylococcus aureus Protein A which is used to construct immuno-amphiphile molecules that assemble into immunofibers in aqueous solution with bioactive epitopes on the surface and have peptide or protein binding ability.

The invention belongs to the fields of gene engineering, bioengineering and biological medicine, and relates to a nucleic acid molecule for coding human defensin, a method for preparing the human defensin and application. The nucleic acid sequence of the nucleic acid molecule comprises any one of the following sequences: (1) a sequence as shown in SEQ ID NO.2; (2) a nucleic acid sequence for coding human defensin, which is obtained by substituting, deleting or adding one or more than two nucleotides to the sequence as shown in SEQ ID NO.2; and (3) a nucleotide sequence which has at least 80% sequence homology with the nucleotide sequence in (1) or (2) and has the same or similar functions with the nucleotide sequence in (1) or (2). According to the method provided by the invention, the recombinant expression vector is constructed by using nucleic acid molecules for coding the human defensin, the human defensin hBD-3 can be recombined and expressed, the recombinant fusion protein is released to the supernatant of a culture solution, the protein purification is convenient, the obtained recombinant human defensin has a tissue regeneration function, and a new active molecule is provided for the development of a novel drug.

The invention discloses a preparation method of a high-stability manual buffer solution, which comprises the following steps of: introducing two types of slow-release porous particle structures, namely a first type of particles loaded with acidic or alkaline components and a second type of particles loaded with silver nanoparticles, so that continuous slow release and dynamic adjustment of the components in the buffer solution are realized; the pH drift and the aging of a buffer system are effectively delayed; meanwhile, slow release of the silver nanoparticles endows the buffer solution with lasting antibacterial protection, microbial growth is remarkably inhibited, and due to the structural design, the long-term pH stability and storage safety of the buffer solution are remarkably improved, the clarity and physicochemical properties of the buffer solution are guaranteed, and the stability of the buffer solution is improved. The method greatly expands the application range in the fields of high-requirement biological experiments, medical detection, long-term sample storage and the like, the particle addition amount and the component proportion can be flexibly adjusted according to actual requirements, the method adapts to different pH ranges and application scenes, and various experiment requirements of protein purification, long-term sample storage, high-sensitivity analysis and detection are met.

The invention discloses a method for separating and purifying bovine lactoferrin by using a ligand affinity chromatography technology, and belongs to the technical field of protein purification. The method comprises the following steps: introducing a bovine lactoferrin gene into pichia pastoris, fermenting to obtain a pichia pastoris fermentation solution, centrifuging, resuspending, homogenizing at high pressure, centrifuging and filtering the pichia pastoris fermentation solution to obtain a filtrate, separating and purifying bovine lactoferrin in the filtrate by using an affinity chromatography technology, immobilizing a triazine dye ligand in a filler of a chromatographic column, and purifying the triazine dye ligand to obtain the bovine lactoferrin. An eluent is prepared, the concentration of NaSCN in the eluent is 0.2-0.5 mol / L, the volume concentration of ethylene glycol in the eluent is 15%-20%, the concentration of NaCl in the eluent is 1-2 mol / L, and the bovine lactoferrin in the chromatographic column is eluted. According to the method, the composition of the eluent is optimized, the three components in the eluent are matched with one another and have a synergistic effect, the stability of the bovine lactoferrin is protected, the purity of the bovine lactoferrin is guaranteed, meanwhile, the elution efficiency of the bovine lactoferrin is improved, and the dosage of the eluent is reduced.

The invention relates to a preparative protein electrophoresis purification system and a protein purification method. The system comprises an automatic gel preparation device and an electrophoresis device, the automatic glue making device comprises a supporting frame, a clamping arm and a glue filling needle, and a linear module is fixedly arranged on the supporting frame to drive the glue filling needle to synchronously move in the horizontal direction and the vertical direction. The electrophoresis device comprises an outer electrophoresis tank, an electrophoresis frame and a cooling device, the electrophoresis frame and the cooling device are arranged in the outer electrophoresis tank, an inner electrophoresis tank is arranged in the electrophoresis frame, rubber plates are detachably arranged on the two sides of the electrophoresis frame, the rubber plates form side plates of the electrophoresis frame, notch grooves are formed in the inner sides of the tops of the rubber plates, and the rubber filling needles are matched with the rubber plates. The protein purification method is a method based on the protein electrophoresis purification system.

The invention discloses an AI-assisted protein purification result analysis method and system. The method comprises the following steps: S1, collecting chromatogram data, electrophoresis image data and mass spectrum peak table data; s2, performing data preprocessing after the chromatogram data, the electrophoresis image data and the mass spectrum peak table data are obtained; s3, carrying out AI identification analysis on the preprocessed data to realize electrophoretic band identification, chromatographic peak identification and mass spectrum deconvolution; s4, performing multi-modal fusion on the chromatography, gel electrophoresis and mass spectrum information sources; and S5, automatically generating a report, and producing a visual chart. The deep learning and multi-modal data fusion technology is introduced, comprehensive automatic analysis of chromatographic data, electrophoresis images and mass spectrum results is achieved, then automatic result analysis of the protein purification process is achieved, an artificial intelligence algorithm is used for recognizing a peak structure and an electrophoresis band, multi-modal data fusion is achieved, and the detection accuracy is improved. And generating a visual analysis report according to a data result.

The invention discloses a construction method and application of a chlamydomonas reinhardtii-sourced high-sweetness protein variant engineering algal strain, the construction method comprises the following steps: carrying out gene modification on sweeteners Thaumatin and Brazzein, optimizing preference codons of chlamydomonas reinhardtii to synthesize CrThaumatin and CrBrazzein encoding genes, cloning the encoding genes into an expression vector pGM6, introducing the recombinant expression vector into a chlamydomonas reinhardtii wild-type chlamydomonas sp. Algal strain, and carrying out high-sweetness protein variant engineering algal strain of the chlamydomonas reinhardtii-sourced high-sweetness protein variant engineering algal strain of the chlamydomonas reinhardtii. And screening by using a paromomycin resistance marker carried by the expression vector to successfully obtain an engineering algal strain. According to the present invention, the engineering strain is subjected to fermentation production to obtain the dry and stable Chlamydomonas reinhardtii powder rich in the target sweet protein, and the protein purification oral test results show that the sweet taste of the Chlamydomonas reinhardtii powder is 3-5 times of the sweet taste of the natural Thaumatin and Brazzein protein;

The utility model discloses a multi-channel protein purification instrument, and relates to the technical field of organic chemical engineering. The purification instrument comprises a purification instrument body, placing plates are arranged on the two sides of the front end of the purification instrument body through moving assemblies, placing assemblies are further arranged on the placing plates, two sets of mounting frames in the moving assemblies are fixedly connected to the two sides of the front end of the purification instrument body in the longitudinal direction respectively, and first lead screws are rotationally connected to the interiors of the mounting frames in the longitudinal direction. According to the test tube rack disclosed by the utility model, the mounting frame and the first screw rod in the moving assembly drive the placing plate to move longitudinally, and the second driving motor and the connecting rod drive the lifting plate and the placing plate to lift, so that the problem that the test tube rack is inconvenient to take and place in the prior art is solved; a movable clamping plate is driven to move through a third screw rod and a connecting strip in the placing assembly, and a centrifugal tube is clamped through a fixed clamping plate and the movable clamping plate, so that the problem that the centrifugal tubes with different sizes are inconvenient to fix in the prior art is solved.

Provided is a technique for improving the efficiency of adsorption of a protein onto a carrier in an apparatus equipped with an in-line mixer and a hydrocyclone. An adsorption method disclosed herein is performed using an apparatus equipped with an in-line mixer and a hydrocyclone connected to the in-line mixer. The adsorption method is a method in which a protein is adsorbed onto a carrier provided with a ligand capable of binding to the protein. The adsorption method includes: mixing a first slurry containing a carrier with a second slurry containing a protein using an in-line mixer to prepare a mixed slurry; and adsorbing the protein onto the carrier in the mixed slurry to produce a complex between the protein and the carrier. The mixed slurry is prepared so as to contain the carrier in an amount of 10-25 vol% and the protein in an amount of 5-20 mg per 1 ml of the carrier when the whole amount of the mixed slurry is 100 vol%. The mixing time in the inline mixer is 0.6-60 seconds.

Porous polymer particles include a polymer matrix and a pore dispersed in the polymer matrix. The pore has a diameter ranging from 50 nm to 500 nm; An epoxy content contained in 1 g of the porous polymer particles ranges from 500 μmol to less than 5000 μmol. Also provided is a column for protein purification including porous polymer particles.

The invention discloses a method for purifying recombinant protein. The framework of the fusion protein is characterized in that a series tag consisting of elastin-like protein (ELP) and SUMO is connected with a target protein. The fusion protein is purified by utilizing the reversible phase change cycle (ITC) property of ELP, and the separation and purification of the recombinant protein can be realized only through simple centrifugal operation, so that the fusion protein with higher purity is obtained, and the required recombinant target protein can be obtained subsequently through enzyme digestion and ITC. The technology is wide in application range, simple and convenient in purification method, low in cost and suitable for industrial amplification, and has a good commercial prospect.

The invention relates to the technical field of protein purification, in particular to a purification method of label-free A53T mutant alpha-synuclein. The purification method disclosed by the invention is a purification method which is more efficient and economical and can maintain protein functions, 20mg of protein can be obtained by inducing 1L of bacterial liquid for 4 hours through IPTG, and the method can improve the overall effect and experimental accuracy of preparation of the label-free A53T alpha mutant-synuclein monomer.

The present disclosure provides a fusion protein comprising a peptide ligand domain that binds to an exosome, a protein purification tag domain, and a water solubility enhancing tag domain. The invention also provides application of the fusion protein in enrichment, capture and / or separation of exosomes. By adopting the fusion protein disclosed by the invention, high-purity and high-activity exosomes can be rapidly, efficiently and specifically captured from cell supernate.

The invention discloses preparation and application of grouper enterospora sporowall protein SWP26 and a polyclonal antibody of the grouper enterospora sporowall protein SWP26, and belongs to the field of animal quarantine, the grouper enterospora sporowall protein SWP26 is obtained by amplifying grouper enterospora genes by using a PCR (Polymerase Chain Reaction) method, shearing a target band and transferring the target band into a pET-32a vector to construct a recombinant plasmid, and then transforming escherichia coli BL21 (DE3) for induced expression. A Ni-NTA affinity chromatography method is used for protein purification, animal immunization and polyclonal antibody purification are carried out on the purified SWP26 recombinant protein, western blot detection is carried out on the purified antibody, an obvious signal appears at about 43kDa, and it is proved that the anti-SWP26 polyclonal antibody can be subjected to a specific reaction with the SWP26 purified protein. The invention clones and identifies the high-abundance spore wall protein SWP26 positioned on the surface of the enterosporidium of the grouper for the first time, belongs to a specific protein of the enterosporidium of the grouper, and can be used as a drug target for treating enterocytozoonosis of the grouper.

Methods and systems for automated protein purification are disclosed. The disclosed methods and systems utilize a plurality of magnetic bead chemistries and an automated extraction system for the purification of one or more proteins within a short time frame.

The instant invention relates to compositions comprising a protein, e.g., an antibody, or antigen-binding portion thereof, e.g., an anti-IL-1R1 antibody or antigen binding portion thereof, and methods, e.g., cell culture and protein purification methods, for producing such compositions.

The invention discloses a method for purifying recombinant X VII type collagen expressed by pichia pastoris fermentation, and relates to the technical field of protein purification methods. The purification method comprises the following steps: centrifuging a recombinant X VII type collagen fermentation liquid fermented and expressed by pichia pastoris engineering bacteria, filtering the obtained centrifugate, then carrying out ultrafiltration concentration, cooling the obtained ultrafiltration concentrated liquid to 2-10 DEG C, then adding Triton X-114, maintaining the material liquid at 2-10 DEG C, carrying out chromatography, leaching and extraction, carrying out ultrafiltration concentration on the extraction liquid, and then carrying out freeze drying to obtain the recombinant X VII type collagen. The recombinant X VII type collagen freeze-dried powder is obtained. The purification method provided by the invention is simple and convenient in whole operation process, can effectively remove endotoxin, can prepare the high-purity recombinant X VII type collagen freeze-dried powder, and is convenient for industrial large-scale application.

The invention provides industrially produced high-purity recombinant human albumin as well as a preparation method and application thereof. Only three-step chromatography is adopted, and high-yield and high-purity recombinant human albumin in gene editing pig plasma can be prepared. Chromatographic packing suitable for protein purification is screened through different purification small-scale experiments, and meanwhile, three types of chromatographic packing are finally confirmed by considering that the requirement for later-stage industrial amplification is met. By regulating and controlling conditions such as sample loading amount, sample loading concentration, pH of samples in different chromatography steps, conductivity value and the like, the recovery rate of albumin is reserved to the greatest extent, the purity of albumin is guaranteed by using three-time chromatography, and the method has the characteristics of high recovery rate, high purity and simplicity in operation, and can be suitable for large-scale preparation of high-purity plasma albumin.

The invention provides a pharmaceutical composition for treating MSS intestinal cancer, and belongs to the technical field of biological medicine, the pharmaceutical composition is used for deeply researching a treatment scheme for effectively inhibiting the MSS intestinal cancer, carrying out basic research from protein purification, proteomics, siRNA libraries and the like, and trying to develop a brand new treatment scheme and a treatment mode using nitixinone, sorafenib and PD1 antibodies; the three medicines are combined for use, so that a remarkable synergistic effect is achieved, and the tumor is successfully changed from cold to hot. The pharmaceutical composition provided by the invention has an important clinical value, breaks through the current difficulty in treatment of MSS intestinal cancer, and opens a brand new direction of application of immunotherapy in clinical advanced metastatic colorectal cancer.

The invention belongs to the field of protein purification, and relates to a method for preparing bevacizumab, which comprises the steps of affinity chromatography, virus inactivation, deep filtration, composite anion exchange chromatography, cation exchange chromatography and the like.

The utility model relates to the field of protein purification, and discloses a protein precipitation supernate drainage device, a precipitation tank is mounted in a mounting cylinder, the upper half part of the precipitation tank is of a cylindrical structure, the inner diameter of the lower end of the precipitation tank is gradually reduced to form a conical part, a pipeline is welded at the bottom end of the conical part, and the lower end of the pipeline is provided with a liquid outlet. A valve is installed below the pipeline, a cover plate is rotationally installed above the centrifugal machine, the cover plate penetrates through the upper portion and the lower portion of the cover plate and is slidably connected with a liquid suction pipe in a sleeving mode, and a liquid suction hose is connected to the end, away from the sedimentation tank, of the liquid suction pipe and connected with a liquid inlet of an external peristaltic pump. The settling tank is fixed in the mounting cylinder, and the settling tank is combined with the centrifugal machine, so that the settling tank can be centrifuged after isoelectric point settling, secondary transfer of protein precipitate after settling is not needed, the loss is reduced, and the convenience is improved.

The utility model relates to a protein high-flux purification device in the technical field of protein purification, which comprises a liquid receiving box, a deep hole plate is sleeved at the upper end of the liquid receiving box, a plurality of deep grooves are arranged at the upper end of the deep hole plate, water outlet holes communicated with the liquid receiving box are arranged on the inner bottom walls of the deep grooves, and a gasket is inserted in the deep hole plate. A pair of rectangular blocks are fixedly connected to a pair of side ends of the deep hole plate respectively; after purification processing, a spray head enters a deep groove, and water is sprayed out of the spray head under the pressure of a rubber plate to flush the inner wall of the deep groove, so that during use, the cleaning steps are simple and time-saving, personnel operation is facilitated, and after cleaning, clean water is sucked into a cover plate to be stored for next cleaning and use; and the deep groove is sealed through the cover plate, so that the interior of the deep groove is not prone to being polluted by dust, and dustproof protection is conducted on the deep groove.